Effects of aflatoxin B1 on the submandibular salivary gland of albino rats and possible therapeutic potential of Rosmarinus officinalis: a light and electron microscopic study.

Background: Aflatoxin B1 (AFB1), a highly toxic mycotoxin, is one of the contaminants of food items such as corn, rice, nuts, and flour. This study aimed to evaluate the effect of AFB1 on the histology and ultrastructure of the submandibular salivary glands (SMSG) of albino rats and examine the possible therapeutic effect of Rosmarinus officinalis extract. Methods: This study used 21 adult male albino rats equally divided into three groups as follows: Group C (saline-treated control group); Group A (AFB1 treated group) subjected to intraperitoneal injection of AFB1 (2 mg/kg) once daily for four weeks; Group R (rosemary-treated group) subjected to AFB1 as in Group A followed by two weeks of intraperitoneal injection of Rosmarinus officinalis extract (400mg/kg) once daily. At the end of the experimental periods, SMSGs were excised and fixed for histological and ultrastructural examinations. Results: SMSGs of the AFB1 group presented atrophied serous acini with numerous cytoplasmic vacuolations; their granular convoluted tubules, striated ducts and excretory ducts presented signs of degeneration in their cell lining with the presence of abundant cytoplasmic vacuolations. In addition, dilated blood vessels engorged with red blood cells were frequently seen. Ultrastructural findings of the AFB1 group showed some acinar cells with degenerated mitochondria presenting loss of cristae and vacuolations as well as irregular, shrunken nuclei with condensed  chromatin. Dilated  rough endoplasmic reticulum were observed in granular convoluted tubules and striated ducts. The glands of animals that received rosemary extract almost regained their normal architecture. Conclusions: It can be concluded that rosemary extract has an ameliorative effect on the deleterious histological and ultrastructural changes induced by chronic AFB1 intake in rat SMSGs.

Microlithiasis in parotid sialadenosis and chronic submandibular sialadenitis is related to the microenvironment: an ultrastructural and microanalytical investigation.

AIMS: Microlithiasis was investigated in parotid sialadenosis and chronic submandibular sialadenitis to determine if it relates to the glandular microenvironment as has been found experimentally. METHODS AND RESULTS: Semithin sections were stained by a mixture of methylene blue and Azure II followed by basic fuchsin, which stains calcified parts of microliths red and organic parts green, and ultrathin sections were examined electron microscopically and microanalytically. Microliths in sialadenosis were found in periacinar stroma, in which necrotic acinar cells were found, and in parenchyma, and consisted of consolidated organic material with little or no crystalline calcium. Microliths in sialadenitis were found in stroma, particularly around intercalary ducts, in lumina and in parenchyma, and contained much crystalline calcium. Macrophages enclosed some microliths. CONCLUSIONS: The paucity of calcium in microliths in sialadenosis and the abundance in sialadenitis relates to the glandular calcium. The periacinar distribution of microliths in sialadenosis possibly relates to formation in periacinar necrotic debris. The distribution of microliths in sialadenitis around intercalary ducts possibly relates to formation in matrix vesicles formed from atrophic parenchyma, and in lumina to formation in stagnant secretory material. Microliths appear to be scavenged by macrophages. Thus the experimental finding that salivary microlithiasis relates to the microenvironment pertaining in humans.

A polarized salivary cell monolayer useful for studying transepithelial fluid movement in vitro.

There are no reported, convenient in vitro models for studying polarized functions in salivary epithelial cells. Accordingly, we examined three often-used salivary cell lines for their ability to form a polarized monolayer on permeable, collagen-coated polycarbonate filters. Only the SMIE line, derived from rat submandibular gland, had this ability. The SMIE cell monolayer exhibited junctional complexes, with a tight-junction-associated protein, ZO-1, localized to cell-cell contact areas. The Na+/K+-ATPase alpha1-subunit was detected predominantly in the basolateral membranes, while the Na+/H+ exchanger isoform 2 appeared primarily in the apical membranes. Using adenovirus-mediated cDNA transfer, SMIE cells were shown to be capable of routing marker proteins (beta-galactosidase +/- a nuclear targeting signal, alpha1-antitrypsin, aquaporin-1) to appropriate locations. Furthermore, this salivary cell monolayer provided a convenient tool for studying aquaporin-1-mediated, osmotically directed, transepithelial fluid movement in vitro. Thus, SMIE cells appear to be a useful experimental model with which to study some polarized functions in a salivary epithelial cell line.

Effects of oxygen, insulin, and glucagon concentrations on rat submandibular acini in serum-free primary culture.

The objective of these studies was to develop serum-free culture conditions for dissociated acini from rat submandibular glands. Acini were isolated from the submandibular glands of 42-46 d old rats and cultured on reconstituted rat tail collagen containing laminin in 1:1 Ham's F12 and Dulbecco's media, supplemented with BSA, transferrin, insulin, T3, EGF, dexamethasone, retinoic acid, carbamylcholine, and trace elements, and gassed with 50% O2. The acini became partly embedded in the collagen gel and rapidly enlarged throughout the first 22 d of culture, maintaining modest seromucous acinar differentiation, as judged morphologically and by mucin secretion. Parallel cultures then were grown under 20, 35, 50, and 65% O2, and evaluated morphologically and by DNA content. Growth and retention of seromucous acinar characteristics were best with 35% O2, but lipid accumulation and cell death were unacceptably high. A spectrum of concentrations of insulin and glucagon then were tried. With 0.05 micrograms/ml insulin, cellular growth and organization were orderly, lipid accumulations were not excessive, and moderate differentiation was retained through 15 d of culture. With more than 0.1 microgram/ml insulin added to or subtracted from the optimum, the detrimental effects recurred. Addition of sufficient glucagon counteracted the effects of both optimum and excessive concentrations of insulin. We now have achieved an orderly growth of moderately differentiated rat submandibular acini for 15 d in serum-free primary culture.

Histochemical localization and X-ray microanalysis of calcium in the rat submandibular gland: demonstration of possible sites for microlith induction.

Rapidly frozen and freeze-substituted submandibular glands of young female rats were embedded in Epon and processed for histochemical demonstration of calcium with the glyoxal bis (2-hydroxyanil) (GBHA) staining method. GBHA staining of thick Epon sections revealed discrete calcium reactions of moderate intensity in practically every secretory granule but not in other compartments of the acinar cells. The saliva in the excretory duct was also reactive with GBHA and showed a drastic decrease in staining intensity toward the distal segments of excretory ducts with larger diameters. In addition, the duct saliva contained numerous tiny particles that were highly GBHA reactive. Stromal cells and cells lining the excretory duct were totally free of reactions. In the acinar cells, X-ray analysis detected distinct peaks for calcium in secretory granules and smaller ones in the Golgi apparatus, while they were undetectable in the rough surfaced endoplasmic reticulum (RER), implicating post-RER calcium loading in the secretory pathway. Electron-dense deposits in the duct saliva showed distinct peaks both for calcium and phosphorus, though these appeared in the acinar secretory granules and other cytoplasmic regions lacked phosphorus. Our observations thus demonstrated physiological calcium in the intra- as well as extracellular compartments of the submandibular gland, and further confirmed drastic changes in chemical composition along the synthetic and secretory pathways of the saliva, by both histochemical and X-ray microanalytical methods. GBHA staining of calcium combined with X-ray microanalysis is useful for an evaluation of the physiology and histo-pathological changes of the salivary glands associated with initial phases of microliths as well as sialoliths formation.

Ultrastructural and immunohistochemical characterization of submandibular duct cells in culture and modification of outgrowth differentiation by manipulation of calcium ion concentration.

The recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to improve normal and CF submandibular duct cell culture capabilities. The duct is most likely the site of the CF defect in this and other exocrine glands. In a previous report conditions required for the successful primary explant culture of normal and CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as severe limiting factors to their utilization in CF research. This report explores the effects of calcium concentrations in the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures. Results of the study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated only after cells proliferate beyond the environment of the explant fragment. Keratinization with subsequent multilayering, desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth medium. Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo ductlike differentiation.

Catalase in salivary gland striated and excretory duct cells. II. phi body: an ellipsoidal peroxisomal organelle with crystalloid axial projections.

A new distinctive and unique peroxisomal organelle with a spindle shape has been observed in luminal epithelial cells of striated and excretory ducts of mouse salivary glands. Light microscopic studies indicate it has an ellipsoidal centre from which catalase-positive filamentous or rod-like processes protrude along its major axis; hence, it is called a phi body. A role for this specialized peroxisome in the formation of nearby free filaments and rods is suggested by the frequent observation of segmentation of its axial processes. Complementary ultrastructural studies of osmium-fixed preparations show that the deformation to an oval shape results from the pressure of the extruding crystalloid coincident with the major axis of the ellipsoidal body. The size range and conformation of phi body axial processes are comparable to those of free catalase-positive rods and filaments observed in the same cells. The periodic substructure of the crystalloid in the phi body core is identical with that of nearby cytoplasmic rods. These observations are consistent with the view that the rods and filaments observed free in the cytoplasm are formed by extrusion from the crystalloid core of the phi body. phi Bodies could also be responsible for the Aver rods of leukemic leukocytes.